PROJECT ABSTRACT Corneal infections caused by the Gram negative bacteria Pseudomonas aeruginosa, the most common cause of contact lens wearing-associated infectious keratitis, and the fungus Candida albicans occur in the USA and worldwide, causing painful, sight-threatening disease. Furthermore, among all human P. aeruginosa infections, more than 13% are multidrug-resistant. This highlights the importance of better understanding the innate immune responses to opportunistic pathogens of the cornea. The objective of this proposal is to defining the protective role of the novel interleukin-36 subfamily of cytokines, close relatives of the IL-1 subfamily, in microbial keratitis and their targets, the dendritic cell. Our preliminary data show that IL-36?, ?, and their receptor IL-36R are required for a proper response to infection, and that down-regulation of their natural inhibitor, IL-36R antagonist, protects the cornea from P. aeruginosa infection. While the IL-1 subfamily is known as one of the most important mediators of innate immunity and inflammation and has been studied extensively with several drugs being developed from this body of research, little is known regarding the role of IL-36R in protective mucosal innate immunity. Based on the preliminary data and published studies, the hypothesis that IL-36R signaling promotes corneal innate defenses against microbial keratitis and antagonizes IL-1? in limiting infection-induced inflammation is postulated and will be tested with three specific aims: 1. To determine the expression and cellular sources of IL-36?, -36? and -36Ra and cell type(s) bearing IL- 36R in the cornea in response to microbial infection. This can be tested by assessing the cellular sources of IL- 36?, ?, and IL-36Ra, and cell types expressing IL-36R in the cornea during murine Pa- and Ca-infection using ELISA of epithelial and corneal extracts, immunohistochemistry. Primary human corneal epithelial cells will be used to assess the means by which IL-36 cytokines are secreted with particular focus on exosomes. The presence of IL-1 cytokines in corneal scrapings from keratitis patients will also be assessed. 2. To decipher the role(s) of IL-36 family members in corneal innate immune responses to infection in vivo and in vitro. This can be tested by assessing the role(s) of IL-36?, ? or IL-36R blockade (using neutralizing antibodies, siRNA, and/or genetic deletions) in mouse models of P. aeruginosa and C. albicans keratitis in vivo and bone marrow derived dendritic cell isolated from wild-type and knockout mice. 3. To assess the therapeutic potential of IL-36? on innate immunity, inflammation resolution, and/or angiogenesis in infected corneas. This can be tested by assessing the effects of topical application of IL-36? in combination with IL-1Ra on the outcome of microbial keratitis in B6 mice. The therapeutic potential of IL-36 stimulated, dendritic cell produced IL-23, and/or ?? T cell produced IL-22 will also be tested. The results from this proposal should improve our understanding of IL-36/IL-36R axis and dendritic cells in the regulation of corneal innate immunity at the molecular and cellular levels and may lead to the development of therapies that limit the progress/pathogenesis of microbial keratitis.